S142: FOXO1-RICTOR AXIS INDUCES ADAPTIVE INCREASE IN AKT ACTIVITY DURING BCR INHIBITOR THERAPY IN CLL: IMPLICATIONS FOR COMBINATION THERAPY
Authors: Laura Ondrisova, Vaclav Seda, Eva Hoferkova, Giorgia Chiodin, Krystof Hlavac, Lenka Kostalova, Daniel Filip, Pedro Faria Zeni, Anna Panovska, Karla Plevova, Sarka Pospisilova, Martin Simkovic, Filip Vrbacky, Daniel Lysak, Stacey M. Fernandes, Matthew S. Davids, Alba Maiques-Diaz, Stella Charalampopoulou, Jose I Martin-Subero, Jennifer R Brown, Michael Doubek, Francesco Forconi, Jiri Mayer, Marek Mraz
Journal: HemaSphere (2023)

Background: Genetic mechanisms of resistance to BCR inhibitors in CLL have been extensively described. However, it remains unknown whether non-genetic adaptation mechanisms to BTK inhibitors might exist. We focused on the possible role of the Akt pathway in adapting to BCR inhibitors since, in mouse models, PI3K-Akt activation is the only known factor that rescues the apoptosis induced by BCR deletion in mature B cells (Srinivasan et al. Cell, 2009). Aims: We aim to describe non-genetic mechanisms of adaptation to BCR inhibitor therapy. Methods: We performed transcriptome profiling (Illumina) and analyzed samples obtained from CLL patients before and during ibrutinib or idelalisib therapy and performed gene editing in MEC1 cells to reveal the functional role of FoxO1/Rictor. Results: We show that in ~80% of CLL cases, ibrutinib increases Akt activity (pAktS473) above pre-therapy levels (34 patients with 104 samples; P<0.005; Fig. A). pAkt was also restored in ibrutinib treated MEC1 cells, where after an initial drop in Akt phosphorylation, its levels were induced within 5 days by >10 folds (P<0.05). Importantly, CLL cells obtained during ibrutinib therapy in vivo were highly sensitive (90% apoptosis) to Akt inhibitor MK2206 (Fig. B). RNA profiling of paired CLL samples obtained before and during ibrutinib (n=22) or single-agent idelalisib therapy (n=18) identified 16 differentially expressed mRNAs (with both drugs) involved in the PI3K-Akt pathway. Rictor induction was particularly notable since it is an essential assembly protein for mTORC2, which is known to phosphorylate Akt directly on S473 (Sarbassov et al. 2005). Analysis of samples obtained during therapy and genome-editing experiments in MEC1 cells revealed that transcription factor FoxO1 is directly responsible for Rictor/pAkt activation during ibrutinib and idelalisib treatment. Knock-out of FoxO1 or Rictor in the MEC1 cell line led to a 70% decrease in pAkt levels (P<0.0001) and resulted in a growth disadvantage in a competitive growth assay in the presence of ibrutinib (Fig. C; P<0.001). Similar results were obtained with idelalisib, suggesting that the FoxO1-Rictor-pAkt axis does not require BTK or PI3Kδ activity. pAkt levels were decreased after combinatory treatment of FoxO1 inhibitor (AS1842856) with ibrutinib or idelalisib in CLL and MEC1 cells. FoxO1 inhibitor decreased basal and ibrutinib-induced pAkt levels and blocked anti-IgM-induced pAkt and cMYC (P<0.01). FoxO1 inhibitor (0.5 uM) induced apoptosis alone (~40% CLL cell killing) or more potently in combination with ibrutinib or idelalisib (~60% apoptosis; Fig. D, n=7). The apoptosis induced by FoxO1 inhibition could not be rescued by coculture of CLL cells with HS5 cells or HS5 engineered to produce CD40L+IL21+IL4, and FoxO1 inhibitor blocked proliferation of primary CLL cells induced by these T-cell factors (Fig. E). Summary/Conclusion: We describe for the first time that the FoxO1-Rictor-pAkt axis is involved in non-genetic adaptation to BCR inhibitors in CLL (see Fig. F) and suggest that FoxO1 is a potential novel therapeutic target since its inhibition decreases CLL cell viability and reduces CLL proliferation induced by T-cell signals. This work was supported by the Ministry of Health of the Czech Rep., grant no. NU20-03-00292 and NU22-03-00117. This research was supported by ERC under the European Union’s Horizon 2020 Research and Innovation Programme (grant agreement no. 802644), National Institute for Cancer Research (Programme EXCELES, ID Project No. X22NPO5102) - Funded by the European Union - Next Generation EU, MH CZ-DRO (FNBr, 65269705), and MUNI/A/1224/2022.Keywords: B-CLL, Resistance, ibrutinib, Bruton’s tyrosine kinase inhibitor (BTKi)